Our goal is to develop tools for subcellular optogenetics. We define subcellular optogenetics as the ability to optically regulate molecular activity in selected regions within a cell. Cells encounter differential concentrations of extracellular stimulus molecules resulting in asymmetric signaling across a cell as in the case of cell migration and neuron differentiation. Specific cellular outputs are thus mediated by signaling activity that is spatially heterogeneous across a cell. Coordinated variations in signaling protein activity are also seen at various sites within the same cell such as the plasma membrane, Golgi or ER which play a key role in regulating cellular functions. To experimentally mimic this spatial variation in activity within a cell and decipher its role in regulating cellular behavior, methods o modulate signaling in selected regions of a single cell are required. We propose to develop such methods specifically with regard to GPCR signaling because it regulates a large variety of critical cellular behaviors such as migration, differentiation, contraction and secretion. Since methods based on genetic or chemical interventions provide limited subcellular control, these methods will be based on optical control of light sensitive molecules. They will build on distinct capabilities we have developed - an opsin based tool to activate GPCRs asymmetrically across a cell and a cryptochrome based tool to generate gradients of activated G protein subunits within a cell. Using these methods, polarized migratory cell behavior can be induced using light. The specific aims will be as follows. Aim 1. To create widely applicable optical tools to specifically inhibit GPCR signaling. Aim 2. To develop optical methods to selectively initiate G protein alpha subunit activity in a cell. Aim 3. To test the hypothesis that to evoke comprehensive immune cell migratory responses, Rac and RhoA need input not only from the G??-PI3K pathway but also an uncharacterized G? pathway. The development of a library of fluorescent protein tools has revolutionized the ability to view subcellular signaling activity in intact cells. A library of optogenetic tools that control this subcellular signaling activity can b transformative in enabling experimental control over cell behavior and identifying its molecular basis. Our goal is to develop such tools by designing light sensitive signaling proteins. They can be used to deduce the role of coordinately regulated variations in signaling activity across a cell The ability to optically localize or create gradients of signaling protein activity within a cell cn be used to regulate critical cellular processes in whole animals.